1. Field of the Invention
This invention relates to the addition of a compound or compounds to a cell-containing fluid sample that permits the sorting and identification of cells based on their distinguishing features without using external energy sources. More particularly, this invention relates to a cell analyzing apparatus and methods for using this apparatus for studying a stream of cells suspended in liquid without the need for an external energy or light source to identify cell or particle distinctions, to quantify such cells or particles, and to separate such cells and particles. Additionally, this invention supplies chemiluminescent compounds usable in such an apparatus with such methods that enable rapid, inexpensive differentiation and quantification of naturally occurring cells and subcellular particles. This invention also relates to the use of the aforementioned compounds for the identification of particular cells under static conditions of cytometry.
2. Description of Related Art
Instrumental analysis of cells can be conveniently divided into four stages: (a) cell preparation, (b) "marking" cells, (c) instrument performance, and (d) data analysis (Schema 1). ##STR1##
Several forms of cells exist in the general circulation of mammals. There are red blood cells which are small ovoid cells without nuclei containing large amounts of hemoglobin which serve to deliver oxygen to peripheral cells and remove carbon dioxide. There are leukocytes, which are larger nucleated cells found, on sedimentation, in the "white" or buffy coat above the red cells. There are at least three populations of leukocytes: (1) a lymphocyte population; (2) a monocyte population; and (3) a granulocyte population which includes neutrophils, eosinophils and basophils. Monocytes contain large nuclei with little cytoplasm. Granulocytes are so named because on staining with hemotoxylin and eosin stains, they are shown to contain cytoplasmic granules. Neutrophils contain non-staining granules, eosinophils contain granules which stain with eosin, and basophils contain granules which stain blue after the administration of an acid wash.
"Marking" of cells comprises either exploiting the physical properties or the biochemical properties of cells.
Cells are distinguished by their physical properties using the Coulter principle disclosed by Coulter Electronics, Inc., Hialeah, FL in U.S. Pat. Nos. 2,656,508, 3,259,842 and 3,603,875. Based upon this principle, cell counting and measuring devices typically employ sensing means that respond to induced changes in electrical resistance or some other electronically measurable parameter in order to count and measure each cell and progressively record cell size and content parameters of a sample of cells suspended in an isotonic electrolyte solution. According to the Coulter principle, when a particle of microscopic size (e.g., a blood cell) passes through an electrical field, there will be a momentary change in the electric impedance in the ambit of the field. The electrical change caused by the passage of a particle through an electrical field of small dimension excited by a direct or low frequency current of high energy is closely proportional to the size and volume of the particle. The electrical pulse in such instruments is produced from an external source.
Using this same basic system, differences in cells created by the composition of the cell can also be amplified. For example, Coulter U.S. Pat. No. 3,502,974 discloses that if the applied current is of sufficiently high frequency, the detectable changes that are produced by the passage of particles are a function, not only of size and volume, but of other physical properties as well, e.g., "opacity" to the electrical field as the result of particle composition. Such qualitative differences can be amplified, detected, then automatically classified.
Identification and separation of normal human leukocytes by volume distribution or other physical characteristics using electrical resistance changes in an applied field, which is the principle of counting and sizing employed by the Coulter Counter instruments, has drawbacks The method is based on the property of all living cells to maintain a certain size and shape. Indeed, each type of cell in the circulating blood has its own characteristic volume ranging from as small as three cubic microns (platelets) to 650 cubic microns for the granulocytes. However, these characteristics are highly variable within the population group with considerable overlap, particularly in diseased states. Advanced Coulter Counters have been designed to make use of such variations in size distribution of platelets, leukocytes, and erythrocytes to detect and monitor pathological states.
Attempts to enhance accuracy have concentrated on alternative means to amplify modest and marginally detectable size differences. Lysing agents have been added to the sheath fluids. Changes in the lysing fluid, which may modify the sizes incrementally, have served to extend the commercial benefits of the basic methodology. These techniques are disclosed in Coulter U.S. Pat. Nos. 4,485,175 and 4,374,644, that disclose the use of particular lysing agents to differentially and gradually modify cell size, and Coulter U.S. Pat. Nos. 4,528,274 and 4,521,518, where the sheath fluid contains charged chemicals that are surface active and thereby enhance charge differences seen between leukocyte populations. Also, see Coulter U.S. Pat. No. 4,555,284 for the use of an osmotic shock agent for a similar purpose.
Devices which make use of combinations of these accuracy enhancing techniques are also known in the art, such as instruments which use a lysing fluid or sheath fluid to modify a physical characteristic such as "electrical opacity" to enhance particle identification, as in Coulter U.S. Pat. No. 3,836,849.
To meet industry needs in this regard, techniques other than ones relying on induced changes in applied electrical fields have been developed to sort and count particular cell populations. But, prior to this invention, no successful method of cell identification or separation using spontaneous light emissions induced by enzymatic cleavage of particular synthetic molecules had been developed.
Until now, the detection by automated instruments of cell differences has required the addition of energy, the scatter, absorption or excitation light reemission of which can be measured by optical or energy sensors. Briefly, as a cell-containing fluid stream passes by an optical or other energy detector, an energy source such as a laser, an electromagnet, or electrical current is directed at the stream. By measuring scattered, absorbed or reemitted energy from the external source, the energy detector or sensor can detect small volume differences or other physical characteristics of the unchanged cell. Additionally, as noted above, the cell can be modified by sheath fluid technology to amplify known physical characteristics. This information is then automatically transmitted to electronically activated deflectors, which can separate the cells by size, or the apparatus can simply count the number of cells within each size category.
Flow cytometers operate on a similar principle. A fluid that contains a known amount of particles per unit volume passes by a sensor. When external energy such as light from a laser, or electromagnetic radiation from an electromagnet, is directed into such a flowing fluid, the particles will scatter, absorb or reemit such energy dependent on characteristics peculiar to such particles. Scattered, absorbed or reemitted energy can be measured by a sensor. The exact amount of such energy received by a sensor per unit time gives a direct indication of the quantity of particles that have passed by in the stream. By knowing the number of such particles per unit volume, the amount of volume per unit time that has passed by can be calculated with an automated instrument--this is, of course, the flow rate Cellular flow cytometers, as in the instant invention, operate by the same principle except that the particles being assayed are cells or subcellular particulate matter, e.g., lysosomes, nuclei, etc.
Sheath fluids used in the standard Coulter technique contain compounds that amplify distinguishing physical features already found in cell populations. Thus, osmotic agents amplify size difference. Paramagnetic agents amplify intrinsic magnetic differences. The sheath fluids used in practicing this invention, however, are radically different from such prior art sheath fluids in that a feature that is not normally automatically detectable, i.e., the presence of a particular species of enzyme or antigen associated with a particular cell population, is made visibly detectable without the need for additional incident energy. In other words, the present invention discloses a technique to make apparent a distinction in the population of whole unseparated cells not normally appreciated by physical instruments.
Generally, prior art devices for detecting cells or distinguishing features of cells required the administration of highly focussed energy, usually electrical, but also electromagnetic radiation, and frequently optically detectable light from a laser or other source. This energy is required because radiation or light detectors or sensors measure scattered, absorbed or reemitted radiation, rather than measuring energy which is spontaneously released or emitted from the cells or particles. Typically, there is no spontaneous emission of radiation from the cells that is usefully counted, sorted, or measured. Any spontaneous energy emission that does occur naturally is either too low in energy level or too infrequently released to be reliably measured by relatively inexpensive instruments. Additionally, induced emissions share these same characteristic difficulties. Thus, added compounds that may produce fluorescence require the administration of external radiation to stimulate optically detectable energy emission. Such an emission is not, therefore, a spontaneous emission within the meaning of the present invention, as fluorescent emissions require the prior or contemporaneous administration of radiation. Additionally, fluorescent radiation is generally of limited temporal duration, which makes it impossible to design a reliable, inexpensive and rapid cell counter based on such emissions. The same principles apply to phosphorescent emissions. Thus, a spontaneous emission that is satisfied by the present invention is measurable by relatively inexpensive instruments, and is of sufficient temporal duration to permit measurement during automatic processing of the fluid containing the cells.
Another means for "marking" individual cells, whether for purposes of static (e.g., microscopic) or flow cytometry, is by cytochemical staining. The principle underlying cytochemical staining is that components of a cell, e.g., a cell surface receptor glycoprotein or an intracellular lysosomal enzyme, can be visualized if one reacts with such component a chemical that becomes colored after reaction (light microscopy) or that fluoresces after reaction by activation by an external light source (fluorescence microscopy). For example, an azo-dye technique for staining monocytes for nonspecific intracellular esterase activity has been combined with computer-assisted flow cytophotometry to assess esterase activity in monocytes suspended in a mixed cell population. Kaplow, L.S., et al., J. Histochem. Cytochem 25:590 (1977). Although this was a marked improvement over classical light microscopic techniques in which it was required that the operator's eyes identify cells based upon form and color, colorimetry is noted for its insensitivity, being limited to about 10.sup.-4 M concentration of the substance detected.
In another example, low receptor numbers on lymphocytes, fibroblasts and epithelial cells were detected by first reacting such receptors with a fluorochrome such as carboxyfluorescein (delivered to the cell covalently linked to protein A, after treating the cells with anti-receptor antibodies), then subjecting the stained cells to fluorescence-based flow cytometry. Dive et al., Cytometry 8:552 (1987). Although fluorescence-based techniques are more sensitive than are colorimetric methods, fluorescence measurements are still limited to about 10.sup.-5 to 10.sup.-6 M concentrations of the compound detected due to high backgrounds and reabsorption of the signal by the fluorophore. Thus, the major disadvantages of dye-based optical methods, whether in the static or flow cytometric modes are: (a) lack of sensitivity, and (b) in the case of fluorescence measurements the requirement for an external energy, i.e., light, source.
Therefore, a great need exists for a method for detecting individual cells that is both ultrasensitive and that obviates the need for any external electrical or energy source.